[Acute promyelocytic leukemia recurrence diagnosed due to granulocytic sarcoma of the external auditory canal].

A 47-year-old man was diagnosed with acute promyelocytic leukemia (APL) accompanied by pancytopenia and left forearm swelling. Complete remission was achieved with remission induction therapy using all-trans retinoic acid (ATRA), and consolidation therapy was completed. Three months after the treatment, left ear closure was observed, and a mass lesion was found in the left external auditory canal. An initial tumor biopsy only revealed inflammatory cell infiltration. Moreover, the tumor's rebiopsy performed 3 months later revealed MPO-positive and CD68-positive granulocyte infiltration. Furthermore, the rebiopsy revealed 4.9×105 copies/µgRNA of PML/RARα, the patient was diagnosed with locally recurrent APL. A bone marrow examination 2 weeks later confirmed an increase in myeloblasts and promyelocytes for the first time since the confirmation of remission. Therefore, it was diagnosed as bone marrow recurrence. Reinduction therapy using ATRA and arsenic trioxide again led to complete remission, after which autologous peripheral blood stem cell transplantation was performed. Currently, complete remission is being maintained. In this case, the recurrence of the external auditory canal lesion preceded the bone marrow recurrence. Therefore, it is important to note the nonspecific leukemia recurrence patterns of the external auditory canal.

IL-7 and IL-15 allow the generation of suicide gene-modified alloreactive self-renewing central memory human T lymphocytes.

Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (T(EM)) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (T(CM)) phenotype. To this, we generated suicide gene-modified T(CM) lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene-modified T(CM) cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene-modified T cells cultured with IL-7 and IL-15 persisted, differentiated in T(EM) cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene-modified T(CM) cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer.

Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.

In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type-specific expression of the Epo gene.

[Successful treatment with rituximab in a patient with refractory thrombotic thrombocytopenic purpura refractory to plasma exchange].

We report a patient with refractory idiopathic thrombotic thrombocytopenic purpura (TTP) who was successfully treated with rituximab. A 50-year-old woman was referred to our hospital with progressive psychoneurotic symptoms, hemolytic anemia and thrombocytopenia. The diagnosis of TTP was confirmed by the absence of ADAMTS13 activity with the presence of circulating ADAMTS13 inhibitor. High-dose steroid therapy and plasma exchange were performed. Despite 21 sessions of plasma exchange, however, there was no remarkable improvement. We then administered rituximab. Fifteen days after the first infusion of rituximab, she achieved complete remission and ADAMTS13 activity increased up to 14%. The patient has remained in remission for more than 9 months.

Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells.

Erythroblast enucleation is thought to be largely dependent on signals mediated by other cells, such as macrophages. In an attempt to improve the in vitro production of red blood cells (RBCs) from immature hematopoietic progenitor cells, we have developed a method to produce enucleated RBCs efficiently in the absence of feeder cells. Our method may represent an efficient way to produce transfusable RBCs on a large scale from hematopoietic progenitors.

Lipocalin 2-mediated growth suppression is evident in human erythroid and monocyte/macrophage lineage cells.

Lipocalin 2 (LCN2), a secreted protein of the lipocalin family, induces apoptosis in some types of cells and inhibits bacterial growth by sequestration of the iron-laden bacterial siderophore. We have recently reported that LCN2 inhibits the production of red blood cells in the mouse. Here we analyzed the role of LCN2 in human hematopoiesis. Expression of LCN2 was observed not only in mature cells such as those of the granulocyte/macrophage and erythroid lineages but also in hematopoietic stem/progenitor cells. We also examined expression of two candidate receptors for LCN2, brain type organic cation transporter (BOCT) and megalin, in various cell types. BOCT showed relatively high levels of expression in erythroid and hematopoietic stem/progenitor cells but lower levels in granulocyte/macrophage and T lymphoid cells. Megalin was expressed at high levels in T lymphoid and erythroid cells but at lower levels in granulocyte/macrophage lineage cells. LCN2 suppressed the growth of erythroid and monocyte/macrophage lineages in vitro, but did not have this effect on cells of other lineages. In addition, immature hematopoietic stem/progenitor cells were not sensitive to LCN2. These results demonstrate a lineage-specific role for LCN2 in human hematopoiesis that is reminiscent of its effects upon mouse hematopoiesis and strongly suggest an important in vivo function of LCN2 in the regulation of human hematopoiesis.

Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo.

Mammalian cells have an activity of mutagenic repair for DNA double-strand breaks (DSBs), microhomology-mediated end joining (MMEJ), in which DNA ends are joined via microhomologous sequences flanking the breakpoint. MMEJ has been indicated to be undertaken without Ku proteins, which are essential factors for non-homologous end joining (NHEJ). On the other hand, recent studies with cell-free (in vitro) systems indicated the involvement of Ku proteins in MMEJ, suggesting that MMEJ could be also undertaken by a Ku-dependent pathway. To clarify whether Ku proteins are essential in MMEJ in vivo, linearized plasmid DNAs with microhomologous sequences of 10bp at both ends were introduced as repair substrates into Ku80-proficient and Ku80-deficient CHO cells, and were subjected to MMEJ and NHEJ. Activities of MMEJ and NHEJ, respectively, of the cells were evaluated by mathematical modeling for the increase in fluorescence of GFP proteins produced from repaired products. The Ku80 deficiency caused approximately 75% reduction of the MMEJ activity in CHO cells, while it caused is > or =90% reduction of the NHEJ activity. Therefore, it was indicated that there is a Ku-dependent pathway for MMEJ; however, MMEJ is less dependent on Ku80 protein than NHEJ. The fraction of MMEJ products increased in proportion to the increase in the amounts of substrates. The results suggest that the increase in DSBs makes the cell more predominant for MMEJ. MMEJ might function as a salvage pathway for DSBs that cannot be repaired by NHEJ.

A new red fluorescent protein that allows efficient marking of murine hematopoietic stem cells.

BACKGROUND: Genetic marking of hematopoietic stem cells (HSCs) with multiple fluorescent proteins (FPs) would allow analysis of their features, including interaction with adjacent cells. However, there are few red FPs that are comparable to green FPs in terms of low toxicity and high fluorescent intensity. This study has evaluated the usefulness of Kusabira Orange (KO) originated from the coral stone Fungia concinna as a red FP for marking of HSCs METHODS: A vector used was the MSCV-type retroviral vector, D Delta Nsap that has the PCC4 cell-passaged myeloproliferative sarcoma virus derived long terminal repeat devoid of a binding site for YY1 and the primer-binding site derived from the dl587rev, respectively. The vector was cloned with the codon-optimized KO cDNA for higher expression in mammalian cells (huKO) and converted to the corresponding retroviruses pseudotyped with the vesicular stomatitis virus G envelope protein, then transduced into c-KIT(+)Sca-1(+)Lineage(-) cells obtained from C57BL/6 (Ly5.1) mice followed by transplantation into lethally irradiated Ly5.2 mice. RESULTS: Approximately 70% of donor-derived cells highly expressed huKO at 16 weeks post-transplantation. Furthermore, the high expression of huKO was also detected in serially transplanted mice, suggesting that expression of huKO per se had little deleterious effect on murine hematopoiesis. In double marking experiments, huKO-expressing hematopoietic cells were easily distinguished from those expressing EGFP by flow cytometry and fluorescent microscope analysis. CONCLUSIONS: Overall, the results obtained from the present study suggest that huKO can be used as a valuable and versatile red fluorescent marker for HSCs.

Detection of the STAT5B-RARA fusion transcript in acute promyelocytic leukemia with the normal chromosome 17 on G-banding.

Acute promyelocytic leukemia (APL) is characterized by chromosomal rearrangements of 17q21, leading to fusion of the gene-encoding retinoic acid receptor alpha (RARA) with a number of alternative partner genes. Signal transducer and activator of transcription 5 beta (STAT5B) is one of the alternative partners. We report a rare case of APL with STAT5B-RARA fusion transcript and the normal chromosome 17 on G-banding. Administration of all trans-retinoic acid improved disseminated intravascular coagulation without decrease of the leukemia cells in his peripheral blood and bone marrow. The molecular mechanism of fusion between STAT5B and RARA by chromosomal rearrangement is discussed based on the data from genome database. Clinical characteristics of APL with STAT5B-RARA are also discussed.

Stable transgene expression in mice generated from retrovirally transduced embryonic stem cells.

Silencing of transduced genes hampers production of transgenic mice using retroviral vectors. We show stable expression of the enhanced green fluorescent protein (EGFP) gene in chimeric mice generated from retrovirally transduced embryonic stem cells. The vector was a murine stem cell virus-typed retroviral vector (GCDsap) in which the long terminal repeat and primer-binding site were derived from a PCC4 cell-passaged myeloproliferative sarcoma virus and the endogenous retrovirus dl587rev, respectively. To increase the viral titer, the vector was packaged with vesicular stomatitis virus G protein, which allowed concentration of the virus into pellets followed by resuspension in serum-free medium. In chimeric mice, EGFP was detected in various tissues including hematopoietic cells, neurons, cardiac muscle, and intestine. Furthermore, high expression was maintained in the progeny of these mice, suggesting successful germline transmission of active proviruses. Although the proportion of EGFP-expressing cells and the mean intensity of EGFP expression varied among tissues and mice, 100% of peripheral blood leukocytes expressed EGFP in mice carrying a single provirus copy, as well as in their progeny. Therefore, the gene transfer system described here provides a useful tool not only to generate transgenic animals but also to manipulate human embryonic stem cells..

L-arginine administration reverses anemia associated with renal disease.

Recombinant human erythropoietin (rhEpo) has proved to be remarkably safe and effective for the treatment of anemia. Despite the use of rhEpo, concerns about its cost, the need for frequent parenteral administration, and the development of anti-Epo antibodies have prompted the development of improved agents to rescue anemia. Patients with anemia associated with renal disease are usually treated by intravenous or subcutaneous rhEpo administration; however, some patients do not respond well to rhEpo, because of the presence of Epo antibody or other unknown reasons. A new, orally administered drug is needed as an economical and effective method to treat such patients. We administered 1.3 g/day of L-arginine to 8 elderly patients with anemia associated with renal disease. All 8 patients responded to the treatment with increases in hemoglobin levels. Six of the patients showed improved renal function. There were no significant adverse effects. Our data show that oral administration of 1.3 g/day of L-arginine significantly improves Epo production and reverses anemia without adverse effects in elderly patients who have anemia associated with renal disease and are in the predialysis state of chronic renal failure.

Long-lasting in vitro hematopoiesis derived from primate embryonic stem cells.

OBJECTIVE: Induction of hematopoietic cells from human embryonic stem (ES) cells has been reported recently. However, before cells derived from human ES cells can be used in the clinic, preclinical studies using these cells in experimental primates will be necessary. Therefore, we attempted to establish a method to induce hematopoietic cells robustly and abundantly from primate ES cells. METHODS: A primate ES cell line, CMK-6, derived from the cynomolgus monkey was used in this study. We adapted a method to induce hematopoiesis from CMK-6 cells on feeder cells, and tested the effectiveness of three kinds of feeder cell lines (OP9, C2C12, and C3H10T1/2). In addition, we tested the effect of vascular endothelial growth factor (VEGF) and insulin-like growth factor-II (IGF-II) on hematopoiesis induction from CMK-6 cells. RESULTS: VEGF and IGF-II showed an extremely strong synergistic effect to induce hematopoiesis from CMK-6 cells. C3H10T1/2 cells proved to be very useful for the induction of hematopoiesis from CMK-6 cells, and the production of blood cells on C3H10T1/2 cells has been maintained as long as 5 months. During this long period, ES cell derivatives continuously produced mature blood cells, including terminally differentiated cells. CONCLUSION: We have developed an original method to produce enriched blood cells abundantly from primate ES cells for an extremely long period. This method may represent a good in vitro model for studying primate hematopoiesis and related diseases. Furthermore, our method may be useful for preclinical studies of transfusion therapy using blood cells derived from ES cells in experimental primate systems.

Lipocalin 2 functions as a negative regulator of red blood cell production in an autocrine fashion.

Members of the lipocalin protein family are typically small, secreted proteins that possess a variety of functions. Although the physiological role of lipocalin 2 remains to be fully elucidated, a few pivotal functions have recently been reported, e.g., regulation of the apoptosis of leukocytes. Unexpectedly, lipocalin 2 is abundantly expressed in erythroid progenitor cells. An in vitro culture experiment demonstrated that lipocalin 2 induces apoptosis and inhibits differentiation of erythroid progenitor cells. During acute anemia the expression of lipocalin 2 was reduced in erythroid cells by a feedback system. Furthermore, injection of recombinant lipocalin 2 into mice suffering from acute anemia retarded the recovery of red blood cell (RBC) numbers, suggesting the importance of reduced expression of lipocalin 2 for the efficient recovery of RBC numbers. These results indicate that lipocalin 2 suppresses RBC production in an autocrine fashion. Hence, anemia arising from pathological conditions, such as chronic inflammation, might be partly due to increased levels of lipocalin 2 secreted from expanded leukocytes and/or macrophages. Also, anemia arising from malignancies might be partly due to the abundant secretion of lipocalin 2 from tumor cells. Thus, lipocalin 2 may represent an attractive therapeutic target for anemia under certain pathological conditions.

Successful treatment of a patient with POEMS syndrome by tandem high-dose chemotherapy with autologous CD34+ purged stem cell rescue.

A 36-year-old man was admitted because of numbness and muscle weakness in the lower extremities. He had gait disturbance, malaise, and body weight loss. Based on the existence of monoclonal gammopathy, the proliferation of abnormal plasma cells in the bone marrow, the presence of sclerotic bone lesion, polycythemia, mild splenomegaly, and an elevated level of serum vascular endothelial growth factor (VEGF) (14,900 pg/mL; normal, 62-707), he was diagnosed as having peripheral neuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome. Following 2 courses of conventional chemotherapy with doxorubicin and dexamethasone, peripheral blood stem cells were mobilized by high-dose etoposide (500 mg/m(2) x 3 days) and granulocyte colony-stimulating factor. After purging by CD34+ selection using the CliniMACS device, the selected cells (12.4 x 10(6)/kg) were cryopreserved. He was then treated with tandem high-dose chemotherapy (HDC) (melphalan 100 mg/m(2) x 2 days) with autologous stem cell rescue. After the first course of HDC, the serum level of VEGF normalized and the minimal residual disease in the bone marrow was reduced below the detection limit of CDR3 analysis by polymerase chain reaction. The patient has been in remission for more than 20 months. He has gradually recovered from the neurological symptoms and now has no impairments of daily living. Our experience suggests that autologous purged stem cell transplantation should be considered as the treatment of choice for POEMS syndrome.

Therapy-related acute myeloid leukemia 6 years after clonal detection of inv(11)(q21q23) and MLL gene rearrangement.

Results of recent studies with animal models suggest that expression of MLL fusion proteins promotes acute leukemogenesis. However, the most potent MLL fusion proteins are not sufficient for the development of acute myeloid leukemia (AML). The clinical data on the pathogenesis of this type of leukemia are limited. We analyzed the case of a patient with therapy-related AML with MLL rearrangement. The patient initially developed AML with t(8;21). Although the patient achieved complete remission with chemotherapy, an abnormal karyotype, inv(11)(q21q23), was detected. After 6-year persistence of a clone with the inversion 11 karyotype in the bone marrow, secondary AML developed. Results of fluorescence in situ hybridization analysis combined with magnet-activated cell sorting analysis showed that MLL rearrangement was detected in CD34+ and CD13+ fractions but not in a CD3+ fraction of the bone marrow. There were 2 important clinical findings. One was that MLL rearrangement was not sufficient for the development of leukemia. The other was that MLL rearrangement targets specific lineages.

Refinement of cytokine use in the in vitro expansion of erythroid cells.

Blood transfusion is indispensable for many clinical applications. However, the supply of transfusable material is insufficient in many countries. Human cord blood contains many hematopoietic stem and progenitor cells, providing a promising resource for the production of transfusable material in vitro. In this study, we have refined a protocol to produce abundant red blood cells (RBC) from human cord blood in an in vitro culture system. We found that erythropoietin and interleukin-3 were most effective when they were added to the culture medium sequentially rather than simultaneously. Although insulin-like growth factor-I (IGF-1) has been reported to function as a positive regulator of RBC production in some in vitro culture systems, we found that IGF-1 had a negative effect upon RBC production. However, IGF-II appeared to function as a positive regulator of RBC production. Finally, stem cell factor functioned to both expand and accelerate the differentiation of immature erythroid cells.

An in vivo assay for retrovirally transduced human peripheral T lymphocytes using nonobese diabetic/severe combined immunodeficiency mice.

OBJECTIVE: Availability of a mouse model to analyze human peripheral lymphocytes genetically modified with retroviral vectors would be useful in T-cell-directed gene transfer studies. To address this issue, we assessed the ability of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to maintain such cells in their peripheral blood. MATERIALS AND METHODS: Human peripheral lymphocytes stimulated with recombinant human interleukin-2 (rhIL-2) and anti-CD3 and CD28 antibodies were transduced with the enhanced green fluorescent protein (EGFP) gene using the retroviral vector GCsap(MSCV) and then transplanted into NOD/SCID mice at 1 x 10(8) cells per mouse. RESULTS: Transplanted human peripheral lymphocytes survived and expressed EGFP in the mice over the 6- to 8-week posttransplant period without any signs of graft-vs-host disease. Of importance was that these cells remained at the G(0)/G(1) stage and again proliferated in response to cytokines when cultured in vitro. Interestingly, the mice in which the transduced T lymphocytes remained at the resting stage clearly elucidated the superiority of the murine stem cell virus (MSCV) LTR to maintain the transgene expression by nonproliferating T lymphocytes over the Moloney murine leukemia virus (MoMLV)- and myeloproliferative sarcoma virus (MPSV)-derived LTRs, which was obscure in in vitro culture where the transduced lymphocytes was being stimulated with rhIL-2. CONCLUSIONS: The mouse model and GCsap(MSCV) vector described herein comprise a simple and reliable in vivo assay system for studies of gene and cell therapies employing human peripheral lymphocytes.

Overproduction of platelet microparticles in cyanotic congenital heart disease with polycythemia.

OBJECTIVES: We sought to clarify the role of platelets in the pathogenesis of abnormal coagulation in patients with cyanotic congenital heart disease (CCHD) with polycythemia; we evaluated the production of platelet microparticles (MPs), platelet degranulation and aggregation response, as well as the correlations of these variables with polycythemia. BACKGROUND: A shortened life span and suppressed aggregability of platelets are well known in patients with CCHD. Although platelet MPs are overproduced and play an important role in the coagulation process in various hematologic and cardiovascular disorders, the production of MPs remains to be elucidated in CCHD. We studied 19 patients who had CCHD with polycythemia and 21 age-matched subjects with acyanotic congenital heart disease (ACHD). Flow cytometry, using monoclonal antibodies, showed the presence of MPs as particles positive for the surface antigen (glycoprotein IIb/IIIa) specific to platelets, and platelet alpha-degranulation was recognized as platelets positive for the surface antigen of P-selectin. Platelet aggregation was assessed as the response to adenosine diphosphate (ADP). Relationships between these indexes and hematocrit (Hct) values were also evaluated. RESULTS: Production of MPs correlated positively with Hct and markedly increased at Hct values above 60% in patients with CCHD. Surface P-selectin and the mean platelet volume in patients with CCHD were comparable with those in patients with ACHD. The platelet aggregation response to ADP significantly and negatively correlated with Hct. In two subjects who showed hemoptysis and underwent phlebotomy, MPs were reduced 6 h after the procedure. CONCLUSIONS: Platelet MPs are overproduced in patients who have CCHD with polycythemia, probably due to a high shear stress derived from blood hyperviscosity. Circulating incompetent platelets, which have already been activated, as well as MPs, might play an important role in the coagulation abnormalities identified in such patients.

Characterization of a myeloma patient with a life-threatening hemorrhagic diathesis: presence of a lambda dimer protein inhibiting shear-induced platelet aggregation by binding to the A1 domain of von Willebrand factor.

We have identified a patient with IgD lambda-type multiple myeloma who was characterized by a severe bleeding tendency, especially after puncture of arterial vessels. Both the bleeding time (>25 min) and activated partial thromboplastin time (APTT) were prolonged. To clarify the underlying pathogenesis, we purified the APTT-prolonging activity from the patient's serum. The purified protein was a highly negatively-charged homodimer of the lambda light chain. The lambda dimer protein (M-protein) inhibited ristocetinand high shear-induced platelet aggregation, dependent on platelet glycoprotein Ibalpha (GPIbalpha), but not epinephrine-, collagen-, ADP-, thrombin-, or botrocetin-induced platelet aggregation. The lambda dimer protein inhibited the binding of platelets to immobilized or ristocetin-treated von Willebrand factor (VWF). Furthermore, a 39/34 kD fragment of VWF encompassing the A1 domain specifically bound to the immobilized lambda dimer protein in the presence of ristocetin, suggesting that the lambda dimer protein directly binds to the A1 domain of VWF. To help elucidate the binding site within the A1 domain, binding of ristocetin-treated VWF to the immobilized lambda dimer protein was assayed in the presence of various anti-A1 domain monoclonal antibodies. Based on these data, we conclude that the lambda dimer protein binds to the region of the A1 domain composed of helices alpha3 and alpha4 and thus interferes with VWF-GPIbalpha interaction. The existence of a protein that inhibits high shear-induced platelet aggregation in acquired von Willebrand disease (VWD) has only rarely been reported. The results suggest that the hemostatic function in arteries with high shear force is profoundly disrupted if the binding of GPIbalpha to VWF is abrogated, supporting the relevance of shear-induced VWF interaction with GPIbalpha in the initiation of the hemostatic process.

A GATA-specific inhibitor (K-7174) rescues anemia induced by IL-1beta, TNF-alpha, or L-NMMA.

Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or N(G)-monomethyl-L-arginine (L-NMMA) are increased in patients with chronic disease-related anemia. They increase the binding activity of GATA and inhibit erythropoietin (Epo) promoter activity. In this study, we examined the ability of K-7174 (a GATA-specific inhibitor) to improve Epo production when inhibited by treatment with IL-1beta, TNF-alpha, or L-NMMA. Epo protein production and promoter activity were induced in Hep3B cells with 1% O2. However, 15 U/ml IL-1beta, 220 U/ml TNF-alpha, or 10(-3) M L-NMMA inhibited Epo protein production and promoter activity, respectively. Addition of 10 microM K-7174 rescued these inhibitions of Epo protein production and promoter activity induced by IL-1beta, TNF-alpha, or L-NMMA, respectively. Electrophoretic mobility shift assays revealed that addition of K-7174 decreased GATA binding activity, which was increased with the addition of IL-1beta, TNF-alpha, or L-NMMA. Furthermore, intraperitoneal injection of mice with IL-1beta or TNF-alpha decreased the hemoglobin concentrations and reticulocyte counts. However, the addition of K-7174 reversed these effects. These results raise the possibility of using K-7174 as therapy to treat anemia.